toxins Article Effects of Sodium Silicate Complex against Hemorrhagic Activities Induced by Protobothrops mucrosquamatus Venom Yen-Chia Chen 1,2,3, Tse-Yao Wang 1,2 , Yu-Kai Huang 4,5,6, Kun-Che Chang 7,8 , Min-Hui Chen 9, Chien-Chun Liu 10 , Kuei-Lin Liu 11, Ya-Han Yang 12, David Hung-Tsang Yen 1,2,3 and Ju-Sing Fan 1,2,* 1 Emergency Department, Taipei Veterans General Hospital, Taipei 11217, Taiwan; [email protected] (Y.-C.C.); [email protected] (T.-Y.W.); [email protected] (D.H.-T.Y.) 2 Department of Emergency Medicine, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan 3 Department of Emergency Medicine, School of Medicine, National Defense Medical Center, Taipei 11490, Taiwan 4 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; [email protected] 5 Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan 6 Department of Surgery, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung 80145, Taiwan 7 Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; [email protected] 8 Spencer Center for Vision Research, Byers Eye Institute, School of Medicine, Stanford University, Palo Alto, CA 94304, USA 9 Enkang Clinic, 3F, 88, Baozhong Rd., Xindian Dist, New Taipei 23144, Taiwan; [email protected] 10 Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; [email protected] 11 Faculty of Biotechnology and Laboratory Science in Medicine, School of Medical Technology and Engineering, National Yang-Ming University, Taipei 11221, Taiwan; [email protected] 12 School of Medicine, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Citation: Chen, Y.-C.; Wang, T.-Y.; [email protected] Huang, Y.-K.; Chang, K.-C.; Chen, * Correspondence: [email protected]; Tel.: +886-228-757-628; Fax: +886-228-738-013 M.-H.; Liu, C.-C.; Liu, K.-L.; Yang, Y.-H.; Yen, D.H.; Fan, J.-S. Effects of Abstract: Protobothrops mucrosquamatus poses a serious medical threat to humans in Southern and Sodium Silicate Complex against Southeastern Asia. Hemorrhage is one of the conspicuous toxicities related to the pathology of Hemorrhagic Activities Induced by P. mucrosquamatus envenoming. Previous in vitro and in vivo studies showed that a silica-derived Protobothrops mucrosquamatus Venom. reagent, sodium silicate complex (SSC), was able to neutralize hemorrhagic and proteolytic activities Toxins 2021, 13, 59. https://doi.org/ induced by pit viper venoms, including Crotalus atrox, Agkistrodon contortrix contortrix and Agkistrodon 10.3390/toxins13010059 piscivorus leucostoma. In this study, we validated that SSC could neutralize enzymatic and toxic effects caused by the venom of P. mucrosquamatus. We found that SSC inhibited the hemolytic and proteolytic Received: 10 December 2020 activities induced by P. mucrosquamatus venom in vitro. In addition, we demonstrated that SSC Accepted: 12 January 2021 could block intradermal hemorrhage caused by P. mucrosquamatus venom in a mouse model. Finally, Published: 14 January 2021 SSC could neutralize lethal effects of P.mucrosquamatus venom in the mice. Therefore, SSC is a candidate Publisher’s Note: MDPI stays neu- for further development as a potential onsite first-aid treatment for P. mucrosquamatus envenoming. tral with regard to jurisdictional clai- ms in published maps and institutio- Keywords: snake venom; snakebite; hemorrhage; Protobothrops mucrosquamatus nal affiliations. Key Contribution: Here, we first report that sodium silicate complex protects mice from P. mucrosqua- matus venom-induced intradermal hemorrhage and lethality. These findings suggest that onsite first-aid SSC application to the snakebite wound in the prehospital setting might be beneficial to Copyright: © 2021 by the authors. Li- patients by alleviating the local toxicities of P. mucrosquamatus venom. censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- 1. Introduction tribution (CC BY) license (https:// creativecommons.org/licenses/by/ Envenoming by venomous snakes poses a serious and challenging global health issue. 4.0/). Kasturiratne et al. [1] estimated that at least 421,000 envenomings and 20,000 deaths occur Toxins 2021, 13, 59. https://doi.org/10.3390/toxins13010059 https://www.mdpi.com/journal/toxins Toxins 2021, 13, 59 2 of 11 each year resulting from snakebites. The highest burden exists in South Asia, Southeastern Asia, and sub-Saharan Africa. The World Health Organization (WHO) recently included snake envenoming as a category A disease in its list of Neglected Tropical Diseases [2]. The mainstay therapy of snake envenoming is the intravenous administration of animal- derived antivenoms, including IgG, fragments F(ab) and F(ab0)2. Although antivenom therapy is largely successful in reducing the mortality associated with snake envenoming, the efficacy of antivenom administration against local symptoms has been limited due to rapid development of damage at the bite sites [3–5]. Hemotoxic venom is composed of proteolytic enzymes and mixed compounds. Among the mixtures, snake venom metalloproteinases (SVMP), phospholipases A2 (PLA2s), and serine proteases mainly account for systemic and local toxicities, such as hemorrhage, allergy, edema, necrosis, and even shock via circulatory system disturbance [6–9]. To allevi- ate local toxicity caused by venom, some experimental studies [4,10,11] reported a novel therapy for the early stage of envenomation using chelators to neutralize metals such as Ca2+, Mg2+, Zn2+, which are known co-factors in the active site of hemotoxic enzymes found in snake venom [12–14]. Although DTPA and EDTA, two well-known metal chela- tors, can neutralize the hemotoxic activities, they provide no significant improvement of either reducing tissue necrosis or increasing survival rate after snake envenomation [15]. In addition, EDTA is contraindicated in the presence of electrolyte imbalance, which is not uncommon after snakebite. A study from the National Natural Toxins Research Center (NNTRC, Texas A&M University-Kingsville, Kingsville, TX, USA) described a patented chemical material, sodium silicate complex (SSC), that was able to neutralize the hemotoxic activity, proteolytic activity and hyaluronidase activity in snake venoms from Crotalus atrox, Agkistrodon contortrix contortrix, and A. piscivorus leucostoma [16]. SSC is a modified silica complex (Figure1) that is water-soluble, carries a pH = 13.7 without having corrosive properties, and is very stable at room temperature. A study conducted in NNTRC also demonstrated that SSC alleviates the subcutaneous hemorrhage induced by venom in- jection in a mouse model [16]. These findings explain the capability of SSC to block hemorrhage caused by pit viper venoms. Most importantly, SSC also shows no evidence of neither irritation on human skin nor reproductive defects in male animals [17]. Other func- Toxins 2021, 13, x FOR PEER REVIEW 3 of 12 tions of SSC, such as anti-cancer and anti-virus activities, were studied by a group of Texas State University [18,19]. These studies suggest that SSC may be a safe and beneficial product for human use. Figure 1. Chemical structure of sodium silicate pentahydrate (SSC). Figure 1. Chemical structure of sodium silicate pentahydrate (SSC). Protobothrops mucrosquamatus has a wide geographic distribution including India, Bangladesh, Myanmar, Laos, Vietnam, Thailand, southern China, Taiwan, and Okinawa [20–23]. Because P. mucrosquamatus envenoming can inflict local effects such as pain, edema, hemorrhage, necrosis, and rare compartment syndrome as well as systemic ef- fects including thrombocytopenia, coagulopathy, rhabdomyolysis, acute renal injuries [24], limb disabilities [22] and death [25], the World Health Organization lists P. mu- crosquamatus among the venomous snakes of highest medical importance in Asia [26]. There are several issues related to antivenom therapy that compromise their effective- ness, including delayed administration of antivenoms in rural settings where public health services and health personnel are scarce [27] and limited effectiveness of anti- venom against venom-induced hemorrhage, necrosis and severe soft tissue swelling de- veloping into airway compromise [28]. In addition, subretinal hemorrhage may occur after snake envenoming, even administered with high doses of antivenom [9]. These evidences show that antivenom therapy may be only partially effective in the control of local tissue damage. In light of the evidence for SSC's efficacy against snake venoms of C. atrox, A. contortrix contortrix, A. piscivorus leucostoma, and Naja subfamily species, SSC administration could be a potential onsite first-aid management to reduce the severity of hemorrhage, edema, and even tissue necrosis prior to antivenom administration in the hospital. To better understand if SSC can neutralize the toxic effects of P. mucrosquamatus venom, we studied the neutralizing effects of SSC on the P. mucrosquamatus venom in vitro and the protective effects against the intradermal hemorrhage and lethality induced by P. mucrosquamatus venom in vivo. 2. Results 2.1. In vitro Evaluation of SSC against Hemolytic and Proteolytic Activities Induced by P. mucrosquamatus Venom In a previous study, SSC was able to neutralize hemorrhagic and proteolytic activi- ties of venom from C. atrox,